My laboratory is focusing on the use of molecular methods to monitor residual disease in three clinical settings: 1. Serial monitoring of BCR-ABL expression in peripheral blood leukocytes for patients with CML undergoing allo-transplantation. Despite the success of tyrosine kinase inhibitors (TKIs) in suppressing CML in most patients, patients who are unresponsive to, or develop resistance to TKIs are being treated experimentally with allogeneic stem cell transplants by investigators in the NHLBI and NCI. The ongoing NIH experience indicates that most of these patients can achieve and maintain a complete or near complete molecular remission post transplant. The sensitive quantitative BCR-ABL PCR assay remains an essential clinical and investigational tool for assessing and managing these patients. 2. Assays designed to detect short tandem repeat (STR) microsatellite polymorphisms are used to monitor engraftment and donor chimerism in patients undergoing stem cell allotransplantation at the NIH are also being used to detect the reappearance of (recipient-derived) leukemia, lymphoma, and myelodysplasia in the blood or bone marrow of transplanted patients. Though limited in sensitivity, chimerism assays can be a valuable tool for disease monitoring particularly in hematologic malignancies lacking an alternative sensitive biomarker. 3. The laboratory has explored the usefulness of quantitative-PCR assays directed against tumor-specific immunoglobulin variable region (VDJ) DNA rearrangements to monitor residual disease in the plasma and bone marrow of patients with multiple myeloma. While routine serum and urine electrophoresis were adequate for monitoring the low grade disease responses achieved in the past, the highly active agents currently under study in clinical protocols initiated by investigators from the NCI often reduce tumor mass below the limits of detectability. Since disease eradication is the ultimate goal, additional sensitive assays capable of guiding further manipulations after complete clinical response has been achieved are clearly needed. The most sensitive standard method for monitoring of residual disease currently in use (bone marrow aspirate/biopsy/flow cytometry) is invasive, and can be limited by sampling error and the difficulty in mobilizing intact plasma cells from marrow stroma for analysis. Cell free DNA detected in the circulation as a consequence of plasma cell turnover/apoptosis, or clonal DNA recovered directly from bone marrow tissue (without cell mobilization) could be useful alternative tools for monitoring low level residual disease with time. In proof of principle studies using real time PCR assays to specifically detect tumor VDJ DNA, our recent studies have confirmed that tumor DNA does circulate in the plasma/serum of most patients with untreated multiple myeloma (7 of 9 patients), and these products rapidly disappeared in response to treatment. Plasma VDJ levels, however, were low in many patients (less than 50 copies/ml plasma) at presentation and disappeared rapidly making them less sensitive than routine serum and urine immunofixation electrophoresis and bone marrow flow cytometry in monitoring residual disease after treatment. In a detailed analysis of these patients, we noted a close correlation between the level of circulating VDJ DNA and the number of plasma cells in the circulation at the time of assay. This implies the circulating clonal DNA is predominantly derived from circulating plasma cells and not from tissue plasma cells. Since the detection of the latter is more important in monitoring of residual disease, without major additional increases in sensitivity perhaps achievable using digital PCR or NGS techniques, the value of cell free VDJ DNA in MRD monitoring low level tissue levels of myeloma may be limited. We also have used tumor specific q-PCR VDJ DNA assays to track tumor cells in DNA extracted from bone marrow aspirate slides of myeloma patients after remission indication. Our preliminary studies indicate tissue VDJ levels can be a very sensitive measure of residual disease, however these studies have been hampered by limited DNA recovery from this tissue source. In principle, fixed marrow clot sections, which contain a much larger volume of tissue, are an attractive alternative source for tissue DNA or RNA for our molecular assay, however the formaldehyde routinely used for tissue fixation denatures and fragments RNA and DNA, markedly reducing their potential value for PCR-based assays which require relatively long nucleic acid templates like VDJ products. To address this problem, we are currently investigating alcohol-based methods of fixation as an alternative. These studies focus on developing fixatives and methods which can maximize recovery of intact DNA and RNA in routine specimens without inordinately altering the histochemical and immunohistochemical properties of the fixed specimens. If convenient reagents and methods can be developed and adopted for routine use, they could have significant improve the quality of nucleic acid products available from conventional clinical samples for all sorts of sophisticated molecular analysis.